Rho‐kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults

Abstract Skeletal muscle haemodynamics and circulating adenosine triphosphate (ATP) responses during hypoxia and exercise are blunted in older (OA) vs. young (YA) adults, which may be associated with impaired red blood cell (RBC) ATP release. Rho‐kinase inhibition improves deoxygenation‐induced ATP release from OA isolated RBCs. We tested the hypothesis that Rho‐kinase inhibition (via fasudil) in vivo would improve local haemodynamic and ATP responses during hypoxia and exercise in OA. Healthy YA (25 ± 3 years; n = 12) and OA (65 ± 5 years; n = 13) participated in a randomized, double‐blind, placebo‐controlled, crossover study on two days (≥5 days between visits). A forearm deep venous catheter was used to administer saline/fasudil and sample venous plasma ATP ([ATP]V). Forearm vascular conductance (FVC) and [ATP]V were measured at rest, during isocapnic hypoxia (80% SpO2), and during graded rhythmic handgrip exercise that was similar between groups (5, 15 and 25% maximum voluntary contraction (MVC)). Isolated RBC ATP release was measured during normoxia/hypoxia. With saline, ΔFVC was lower (P < 0.05) in OA vs. YA during hypoxia (∼60%) and during 15 and 25% MVC (∼25–30%), and these impairments were abolished with fasudil. Similarly, [ATP]V and ATP effluent responses from normoxia to hypoxia and rest to 25% MVC were lower in OA vs. YA and improved with fasudil (P < 0.05). Isolated RBC ATP release during hypoxia was impaired in OA vs. YA (∼75%; P < 0.05), which tended to improve with fasudil in OA (P = 0.082). These data suggest Rho‐kinase inhibition improves haemodynamic responses to hypoxia and moderate intensity exercise in OA, which may be due in part to improved circulating ATP. Key points Skeletal muscle blood flow responses to hypoxia and exercise are impaired with age. Blunted increases in circulating ATP, a vasodilator, in older adults may contribute to age‐related impairments in haemodynamics. Red blood cells (RBCs) are a primary source of circulating ATP, and treating isolated RBCs with a Rho‐kinase inhibitor improves age‐related impairments in deoxygenation‐induced RBC ATP release. In this study, treating healthy older adults systemically with the Rho‐kinase inhibitor fasudil improved blood flow and circulating ATP responses during hypoxia and moderate intensity handgrip exercise compared to young adults, and also tended to improve isolated RBC ATP release. Improved blood flow regulation with fasudil was also associated with increased skeletal muscle oxygen delivery during hypoxia and exercise in older adults. This is the first study to demonstrate that Rho‐kinase inhibition can significantly improve age‐related impairments in haemodynamic and circulating ATP responses to physiological stimuli, which may have therapeutic implications.

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----------------EDITOR COMMENTS Reviewing Editor: The study supports a role of RBC rho-kinase in plasma ATP and blood flow, and the potential of Rho-kinase inhibition as an intervention that increases forearm blood flow during intense exercise in old individuals. Data are novel, nicely presented ad discussed, and the study could have a significant impact on the field. The research group has a solid experience in the field.
Both reviewers make precise comments and raise some issues (mainly related to data interpretation and analysis) which should be convincingly addressed by the authors. -----------------

GENERAL COMMENTS
This comprehensive study employed the rhythmic handgrip exercise model in conjunction with in vivo measurements of blood flow, blood oxygenation and plasma ATP in young and older individuals in normoxia, hypoxia and venous fusadil infusion and ex-vivo RBC ATP release in comparable conditions to investigate the impact of RBC rho-kinase inhibition in limb blood flow and circulating ATP. Interestingly, no absolute differences in forearm blood flow (FBF) or plasma [ATP] were observed between the older and young groups at rest in normoxia, at rest in hypoxia or during most of the subpeak rhythmic handgrip exercise bouts with either saline or fusadil infusion. The main positive findings were that systemic venous fusadil infusion induced 1) increases (above baseline) in FBF in hypoxia in both groups and 2) elevations in peak exercise hyperaemia in the older group (compared to baseline), but not in the young individuals. No absolute between group differences in FBF were observed at peak exercise with fusadil infusion. The study appears to have been well-conducted and the results with fusadil infusion and ex-vivo RBC ATP release are novel. That being said, the paper overemphasises the positive results in the peak exercise condition and fails to provide the reader with information about the absolute differences in FBF as well as information about the conditions were FBF was not different between groups. This is important to put in perspective the magnitude of the phenomenon under investigation and thus provide insight into its therapeutic potential. There is also a need to clarify how arterial O2 content and forearm VO2 were estimated to convince the reader of the validity of the reported statistical differences in forearm VO2, which are within the measurement error of the multiple variables used in this calculation. Lastly, it would be helpful to acknowledge in the experimental considerations and limitations section that further work is needed in older people undertaking other exercise modalities (including exercise capacity tests during larger muscle mass exercise) to ascertain the therapeutic potential of pharmacologically induced rho-kinase inhibition.

SPECIFIC COMMENTS
TITLE -Specify that the rhythmic handgrip exercise model was employed. The effects of Rho-kinase inhibition during other exercise modalities remains unknown and thus the results cannot be generalised.
KEY POINTS -the background information tells the reader that a general impairment in skeletal muscle blood flow occurs with exposure to hypoxia and exercise, but this is not supported by the data of the present study, which only showed FBF differences at peak exercise in the saline infusion trial. The general statements in the first and last key points therefore needs to be amended to specify the conditions where blood flow in older people is lower and thus its regulation might be 'impaired' (or altered) compared to young individuals. differences in FBF were only observed at peak exercise in the saline trial. This is crucial to be able to evaluate the potential implications of the study for older people undertaking other ecologically relevant exercise modalities. Statements of the magnitude of difference in FBF between older and young people during exercise and the variability in this and plasma [ATP] measurements are needed to address this concern.
There is little discussion of the negative observations revealing similar between groups FBF at rest (normoxia and hypoxia) or during handgrip exercise at 5%MVC and 15%MVC with either saline or fasudil infusions. Between group FBF differences were only observed at 25% MVC (335 vs. 278 mL/min in older; P<0.05). This effect during the saline condition could be in part confounded by differences in workload (8.1 vs 7.4 kg; FBF per kg = 41 vs. 38 ml/min/kg in young vs. older group). Please clarify whether the normalised data would still be statistically different.
Referee #2: The manuscript by Racine et al describes potential mechanisms to ameliorate age-induced impairments in skeletal muscle vascular function and hemodynamic responses through improvements in circulating ATP during both hypoxia and hand grip exercise. The authors demonstrate that decreased ATP release may be involved in the increased risk of CVD and the decline in functional capacity with primary aging. Further, the use of Fasudil may alleviate age-associated CVD risk and/or the declines in functional capacity. The experimental paradigm is well designed, and the manuscript is well written. However, there are some concerns with the data and analysis and aspects of the discussion that must be addressed. Below are major and minor concerns.

MAJOR CONCERNS
1. In the key points section, you state "...fasudil, improved blood flow and circulating ATP responses during hypoxia and hand grip exercise compared to adults...". However, in Figure 2, this does not appear to be the case during hypoxia. Unless I am missing something, there is no comparison made between older adults (OA) and young adults (YA). figure 2E, OA [ATP]v with fasudil and hypoxia is significantly different versus fasudil and normoxia, however, in table 3, they are not significantly different. With hypoxia and fasudil versus hypoxia and saline in OA, [ATP]v is not different. Further, in figure 2F, hypoxia and fasudil did not increase ATP effluent versus saline and hypoxia in OA. Please explain these discrepancies and lack of significant difference with Fasudil versus saline, as this is clearly a primary outcome.

In
3. Figure 3A-D: How was delta blood flow calculated? Should it not be in %change from rest? As it stands right now, the absolute blood flow values and change in forearm blood flow are nearly the same absolute values; how can this be? 4. In Figure 5, extracellular ATP in isolated RBC from OA was not different versus saline and fasudil treatment. However, you state that fasudil improved isolated RBC ATP release. figure 2E, [ATP]v in YA during hypoxia is significantly different versus saline and normoxia. However, fasudil did not improve [ATP]v with hypoxia versus normoxia and fasudil. Why would fasudil improve the [ATP]v with hypoxia in OA but not YA?

In
6. You showed that fasudil lowered blood pressure and improved forearm blood flow in OA. Fasudil and ROCK inhibition, has been shown to decrease blood pressure and circulating ANG-II, at least in an animal model of hypertension (Ocaranza et al. Hypertension, 2011). Given older adults typically have higher sympathetic activation, and thus likely elevated circulating angiotensin-II (ANG-II). Do you think that these fasudil-mediated changes in ANG-II may be occurring with your data and thus may be a potential mechanism for the hemodynamic improvements in OA?

12-Dec-2021
MINOR CONCERNS 1. Fasudil is a vasodilator, what role do you think this fasudil-mediated vasodilation played in the ATP and hemodynamic responses herein? Is fasudil acting as the vasodilator and increasing ATP release or is fasudil increasing ATP release which then facilitates vasodilation? 2. Should VO2 (ml/min) be normalized to relative VO2 (ml/min/kg) for proper comparison between young and old?
3. This is very minor in my opinion, but consider consolidating the duplicated data presented in the tables and figures.
4. The introduction could be shortened and refined for brevity without losing the core information, and at the very least, more justification for the use of fasudil should be included.

Reviewing Editor:
The study supports a role of RBC rho-kinase in plasma ATP and blood flow, and the potential of Rho-kinase inhibition as an intervention that increases forearm blood flow during intense exercise in old individuals. Data are novel, nicely presented and discussed, and the study could have a significant impact on the field. The research group has a solid experience in the field. Both reviewers make precise comments and raise some issues (mainly related to data interpretation and analysis) which should be convincingly addressed by the authors.
Response: We thank the reviewing editor and referees for their positive comments regarding our study. We have addressed the concerns below, made revisions where appropriate, and believe the revised manuscript is strengthened as a result.

This comprehensive study employed the rhythmic handgrip exercise model in conjunction with in vivo measurements of blood flow, blood oxygenation and plasma ATP in young and older individuals in normoxia, hypoxia and venous fasudil infusion and ex-vivo RBC ATP release in comparable conditions to investigate the impact of RBC rho-kinase inhibition in limb blood flow
and circulating ATP. Interestingly, no absolute differences in forearm blood flow (FBF) or plasma [ATP] were observed between the older and young groups at rest in normoxia, at rest in hypoxia or during most of the subpeak rhythmic handgrip exercise bouts with either saline or fasudil infusion. The main positive findings were that systemic venous fasudil infusion induced 1) increases (above baseline) in FBF in hypoxia in both groups and 2) elevations in peak exercise hyperaemia in the older group (compared to baseline), but not in the young individuals. No absolute between group differences in FBF were observed at peak exercise with fasudil infusion. The study appears to have been well-conducted and the results with fasudil infusion and ex-vivo RBC ATP release are novel. That being said, the paper overemphasizes the positive results in the peak exercise condition and fails to provide the reader with information about the absolute differences in FBF as well as information about the conditions where FBF was not different between groups. This is important to put in perspective the magnitude of the phenomenon under investigation and thus provide insight into its therapeutic potential. There is also a need to clarify how arterial O2 content and forearm VO2 were estimated to convince the reader of the validity of the reported statistical differences in forearm VO2, which are within the measurement error of the multiple variables used in this calculation. Lastly, it would be helpful to acknowledge in the experimental considerations and limitations section that further work is needed in older people undertaking other exercise modalities (including exercise capacity tests during larger muscle mass exercise) to ascertain the therapeutic potential of pharmacologically induced rho-kinase inhibition.
Response: Thank you for your positive comments as well as your suggestions for improvement. We have amended the manuscript accordingly.

TITLE -Specify that the rhythmic handgrip exercise model was employed. The effects of Rhokinase inhibition during other exercise modalities remains unknown and thus the results cannot be generalised.
Response: Good point. Amended as suggested.
KEY POINTS -the background information tells the reader that a general impairment in skeletal muscle blood flow occurs with exposure to hypoxia and exercise, but this is not supported by the data of the present study, which only showed FBF differences at peak exercise in the saline infusion trial. The general statements in the first and last key points therefore need to be amended to specify the conditions where blood flow in older people is lower and thus its regulation might be 'impaired' (or altered) compared to young individuals.
Response: Impaired muscle blood flow and vascular conductance in older vs. young adults have been demonstrated by our laboratory and others during various exercise modalities (e.g., handgrip exercise, knee extensor exercise, cycling exercise) and various exercise intensities ranging from light to high intensity depending on the study (see review by Hearon Jr. & Dinenno, 2016). This is also the case when expressing the haemodynamic data as absolute values or a response from baseline (i.e., change/delta) to the exercise stimulus. Given this, we purposefully kept this statement general because it would be too difficult to specify the exact exercise conditions where this has been demonstrated particularly given the strict word limitations of the Key Points. Regarding the data in the present study, we agree with the Referee and made sure to emphasize "moderate intensity exercise" in the manuscript, as 15 and 25% MVC represent ~40 and ~70% WRmax, respectively (Richards et al., 2014). Also, when the change in haemodynamics from baseline is different between groups of conditions, we use the term "response" to take into account individual differences in baseline blood flow and vascular conductance.
ABSTRACT -State whether the absolute exercise intensities were comparable between groups. As the authors know, this is critical to determine hemodynamic differences between groups, as the metabolic demand determined by the absolute work rate influences the magnitude of blood flow. Similarly, provide quantitative data of the magnitude of blood flow differences between groups to establish the physiological significance of the phenomenon.
Response: There were no differences in absolute exercise workloads and we added this briefly to the abstract. Given the amount of data collected in this study, it is not possible to add much quantitative data given the strict word limitations. Thus, we respectfully have chosen to keep the abstract as a comprehensive descriptive abstract.

ABSTRACT -Is the conclusion relating the improved circulating ATP and blood flow in older group based on any correlational analysis of FBF vs. plasma [ATP]?
Response: We have performed correlational analyses in several prior studies on this topic, and we have found it very difficult to interpret. As the reviewer is aware, venous plasma [ATP] is quite literally a "snapshot" of ATP concentrations at the level of a conduit vein draining the forearm circulation. We cannot measure this at the level of the microcirculation, which would be ideal. This, in combination with our understanding that the half-life of ATP in circulation is less than 0.5 seconds (Mortensen et al., 2011), and is so rapid that infusion of ATP into the brachial artery (at a concentration that causes profound vasodilation) cannot be detected on the venous side of the circulation (i.e., it is broken down in one pass of the circulation in this small tissue model in vivo; Kirby et al., 2012), often results in poor correlations. Thus, we are cautious in emphasizing correlations with plasma ATP and recognize it is difficult to accurately measure (see page 20 of discussion). Mechanistic studies involving blockade of purinergic 2y (P2y) receptors or key signalling pathways downstream of P2y receptor stimulation will be needed to definitively address this issue.
INTRODUCTION -End of the introduction. Clarify why the rhythmic handgrip exercise is used to answer the question of the study.
Response: This was used based on prior observations in our lab for both haemodynamics (Kirby et al., 2012;Richards et al., 2014) and plasma ATP (Kirby et al., 2012). We have also shown impaired haemodynamic and ATP responses to hypoxia in the forearm circulation (Kirby et al., 2012). This has been added per your suggestion.

METHODS -Page 10. The absolute forearm vascular conductance values in this model are microliters/min/mmHg. Why do they need to be expressed per 100 mmHg?
Response: Our lab and others have used this approach to normalize the conductance per 100 mmHg which yields comparable values to blood flow (Tschakovsky et al., 2002).
Page 10. The authors report the weights corresponding to the workloads used in the study in Table 1. Did you also measure the rate of contraction to obtain some index of power? The reported weight data do not seem to indicate that power was the same in both groups, as indicated by the statistical analysis.
Response: Given the similar workload in kg (P-values for the absolute workload comparison between young vs. older subjects at 5%, 15%, 25% MVC were 0.563, 0.463, and 0.499, respectively; individual subject data shown below) and identical load distance (0.035m) and duty cycle (20 contractions per minute) as monitored and performed with both audio and visual cues via metronome as done regularly in our lab, indices of work and power would be similar to the absolute load lifted. Response: Any estimate of arterial O2 content will have some degree of variability and error in the absence of a brachial artery catheter to directly measure arterial blood gases. For example, using pulse oximeter-derived SO2 data in the present study to estimate PO2 for calculating arterial O2 content provides PO2 estimates in normoxia that range from ~80mmHg to >145mmHg, which is far above direct measurements of arterial PO2 taken previously in our laboratory (Racine et al., 2018). Thus, while we agree that all participants would not be expected to have the exact same arterial O2 content, we chose to utilize a consistent estimate of arterial O2 content across subjects based on direct brachial artery blood gas measurements from similar healthy young and older subjects who participated in similarly conducted prior studies within our laboratory (Richards et al., 2014(Richards et al., , 2017; no arterial O2 content differences with age) rather than more indirect estimates based on pulse oximetry. Response: We have added a statement to the results section (page 15, lines 15-18) to include this analysis. This impacts the age-group differences in FBF, but not FVC for the 25% MVC intensity. Importantly however, this normalization does not explain the fasudil-mediated improvements during exercise in older adults, nor the improved responses during hypoxia. While we understand this comment and the suggested normalization, this may reflect more math than physiology, and the proper test of this would require young and older adults performing an exercise at identical absolute workloads. In revision, we have been mindful and cautious in our data interpretation, and have highlighted the novelty of the data as it relates to the fasudil-mediated improvements in the older subjects, as this is the key finding and has never been demonstrated before. Response: These are excellent points, and we have attempted to address these in revision throughout and in the experimental considerations section. Response: Similar FBF and FVC at rest is an expected finding as our lab and others have shown this repeatedly. While the absolute FBF/FVC are not different between groups during hypoxia, the response (Δ) is different and important when considering individual differences in baseline haemodynamics. We addressed the normalisation data above. Importantly, normalising the data does not explain the fasudil-mediated improvements in FBF/FVC responses during hypoxia and moderate intensity exercise in older adults.

Referee #2:
The Response: Thank you for the positive comments and enthusiasm for our study. We have addressed your concerns below and in the revised manuscript.

MAJOR CONCERNS
1. In the key points section, you state "...fasudil, improved blood flow and circulating ATP responses during hypoxia and hand grip exercise compared to adults...". However, in Figure 2, this does not appear to be the case during hypoxia. Unless I am missing something, there is no comparison made between older adults (OA) and young adults (YA).
Response: We did in fact compare the FBF/FVC responses (Δ FBF/FVC) between young and older (e.g., Figure 2C and D) and also with saline or fasudil. This was also done for circulating ATP (ATP effluent, Figure 2F). figure 2E, Response: In Figure 2E, [ATP]v tended to be greater in hypoxia with fasudil vs saline in the older adults (P=0.073). Table 3 shows data from the exercise trials, so we are unclear about this comment as it relates to hypoxia. In Table 2, the data is shown as not significant, but again the P-value was 0.07. Per Referee #1's suggestion, and that it is difficult working back and forth through the figures and tables, we have removed absolute FBF, FVC and [ATP]v from the tables as these are shown in Figures 2 and 3. In Figure 2F, ATP effluent was slightly lower at baseline with fasudil in the older subjects. ATP effluent was significantly increased during hypoxia in older adults within condition (P<0.0001), but this value did not achieve statistical significance compared with that observed at baseline (normoxia) with saline (P=0.251).

In
3. Figure 3A-D: How was delta blood flow calculated? Should it not be in %change from rest? As it stands right now, the absolute blood flow values and change in forearm blood flow are nearly the same absolute values; how can this be?
Response: Delta blood flow was calculated as either hypoxia blood flow or exercise blood flow (for each intensity) minus resting blood flow within condition. There are many ways to express the data. Given that there were no age group differences in resting forearm blood flow (or vascular conductance), we opted to present the absolute change in FBF to account for individual differences. Given that resting FBF is relatively low (~20 ml/min), the values for the change in blood flow are quite similar to absolute blood flow since the increase in FBF is quite large during exercise. Figure 5, extracellular ATP in isolated RBC from OA was not different versus saline and fasudil treatment. However, you state that fasudil improved isolated RBC ATP release.

In
Response: This is based on the comparison within condition. Extracellular ATP was not significantly increased above baseline in response to hypoxia in the saline condition, but was in the fasudil condition ( Figure 5A). We have presented this in a different form in Figure 5B and have described this in the respective paragraph in the results (page 16).

In figure 2E, [ATP]v in YA during hypoxia is significantly different versus saline and normoxia. However, fasudil did not improve [ATP]v with hypoxia versus normoxia and fasudil. Why would fasudil improve the [ATP]v with hypoxia in OA but not YA?
Response: This is the primary hypothesis of the study. We have shown previously that red blood cells from older adults are less deformable and release less ATP during hypoxia than young adults, and treatment in vitro with a rho kinase inhibitor improves deformability and ATP release in older adults only. Thus, we were attempting to translate these findings to an in vivo model. The present data, to some extent, support this hypothesis.
6. You showed that fasudil lowered blood pressure and improved forearm blood flow in OA. Fasudil and ROCK inhibition, has been shown to decrease blood pressure and circulating ANG-II, at least in an animal model of hypertension (Ocaranza et al. Hypertension, 2011). Given older adults typically have higher sympathetic activation, and thus likely elevated circulating angiotensin-II (ANG-II). Do you think that these fasudil-mediated changes in ANG-II may be occurring with your data and thus may be a potential mechanism for the hemodynamic improvements in OA?
Response: Interesting point. This is possible, however we believe this is unlikely as our previous studies demonstrate that the age-related impairments in blood flow responses to hypoxia or graded exercise are not due to elevated sympathetic nervous system activity or greater sympathetically-mediated vasoconstriction (Richards et al., 2014(Richards et al., , 2017. We have not performed studies with ANG-II blockade, but we do not believe this is likely.

Fasudil is a vasodilator, what role do you think this fasudil-mediated vasodilation played in the ATP and hemodynamic responses herein? Is fasudil acting as the vasodilator and increasing ATP release or is fasudil increasing ATP release which then facilitates vasodilation?
Response: Great question, and from the present study design, it is not possible to definitively address this. We don't believe that the possible direct vasodilator effect of fasudil explains the findings for a few reasons. First, baseline FBF and FVC were not significantly different with fasudil vs. saline, and the responses were only different during the hypoxic or exercise stimuli. As for venous plasma [ATP], previous studies from our laboratory and others have shown that vasodilation per se does not increase plasma [ATP] (Mortensen et al., 2011;Kirby et al., 2012), and may actually decrease it due to potential dilution effects of elevated blood flow.
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31-Mar-2022
Michael C.  The study provides novel evidence that Rho-inhibition is associated with increases in limb blood flow and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older individuals. The topic is of interest and both referees expressed positive comments. Whereas Reviewer 2 is satisfied with the revision, Reviewer 1 still has one pertinent comment which the authors should take into consideration. The Reviewer suggests to estimate CaO2 values from data of the present study (and to recognize the limitation associated with the estimate), without taking CaO2 values from a previous study. -----------------

REFEREE COMMENTS
Referee #1: This reviewer appreciates the thorough responses to the comments and the corresponding amendments in the manuscript. The clarity of the manuscript has been improved as the result. There is, however, a remaining concern that needs further consideration, namely the use of CaO2 mean data from a previous study to calculate O2 delivery and forearm VO2 in the present study. A stronger approach is to use the present directly measured individual venous [Hb] and SpO2 values to estimate the CaO2 values for each participant and condition. The corresponding PaO2 can be estimated from a standard O2 dissociation curve. The latter will not have a major impact in CaO2 in comparison to differences in [Hb] and O2Hb among participants and conditions. The fact that this is an estimate should be mentioned in the experimental considerations section of the discussion in particular in relation to the reported findings based on estimates of forearm VO2. Lastly, it is recommended to report [Hb]v in tables 2 and 3, as done for the isolated RBC experiment in Table 4.
Referee #2: Overall, this is an impactful study and is of high-impact to the field. Well done.

END OF COMMENTS
1st Confidential Review 20-Apr-2022 2nd Authors' Response to Referees

EDITOR COMMENTS
Reviewing Editor: The study provides novel evidence that Rho-inhibition is associated with increases in limb blood flow and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older individuals. The topic is of interest and both referees expressed positive comments. Whereas Reviewer 2 is satisfied with the revision, Reviewer 1 still has one pertinent comment which the authors should take into consideration. The Reviewer suggests to estimate CaO2 values from data of the present study (and to recognize the limitation associated with the estimate), without taking CaO2 values from a previous study.
This reviewer appreciates the thorough responses to the comments and the corresponding amendments in the manuscript. The clarity of the manuscript has been improved as the result. There is, however, a remaining concern that needs further consideration, namely the use of CaO2 mean data from a previous study to calculate O2 delivery and forearm VO2 in the present study. A stronger approach is to use the present directly measured individual venous [Hb] and SpO2 values to estimate the CaO2 values for each participant and condition. The corresponding PaO2 can be estimated from a standard O2 dissociation curve. The latter will not have a major impact in CaO2 in comparison to differences in [Hb] and O2Hb among participants and conditions. The fact that this is an estimate should be mentioned in the experimental considerations section of the discussion in particular in relation to the reported findings based on estimates of forearm VO2. Lastly, it is recommended to report [Hb]v in tables 2 and 3, as done for the isolated RBC experiment in Table 4.
Response: We have calculated CaO 2 in this manner per your request, and have added [Hb]v to Tables 2 and 3. We have also included the following statement on page 18 regarding blood gasses: "Given that we estimated CaO 2 in the present study and that blood gasses were not obtained in all subjects, caution should be taken when interpreting the findings related to forearm V̇O 2 ." All changes to the revised manuscript are in red font. We hope you find our manuscript now acceptable for publication, and we appreciate your time, efforts, and critical yet positive feedback on our work.

Referee #2:
Overall, this is an impactful study and is of high-impact to the field. Well done.
Response: Thank you!

28-Apr-2022 2nd Revision -Editorial Decision
Dear Dr Dinenno, Re: JP-RP-2022-282730R2 "Rho-kinase inhibition improves haemodynamic responses & circulating ATP during hypoxia & moderate intensity handgrip exercise in healthy older adults" by Matthew L. Racine, Janée D. Terwoord, Nathaniel B. Ketelhut, Nate P. Bachman, Jennifer C. Richards, Gary J Luckasen, and Frank A. Dinenno Thank you for submitting your revised Research Article to The Journal of Physiology. It has been assessed by the original Reviewing Editor and Referees and has been well received. Some final revisions have been requested.
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